Testicular germ cell tumour arising 15 years after radiotherapy with 18 Gy for germ cell neoplasia in situ.

PURPOSE: Germ cell neoplasia in situ (GCNis), the precursor of adult testicular germ cell tumours (GCTs), is found in 5-6% of contralateral testicles in patients with testicular GCT and in the tumour-surrounding tissue of > 90% of testes undergoing testis-sparing surgery (TSS) for GCT. Local radiotherapy to the testis with 18-20 Gy eradicates GCNis while preserving Leydig cells. The frequency of treatment failures is so far unknown. METHODS: A 22-year-old patient with right-sided seminoma clinical stage I and contralateral GCNis received radiotherapy with 18 Gy to his left testicle. Fifteen years later he underwent orchiectomy of the irradiated testis for seminoma with adjacent GCNis. The patient is well 1 year postoperatively while on testosterone-replacement therapy. The literature was searched for further cases with GCTs arising despite local radiotherapy. RESULTS: Six failures of radiotherapy have been reported previously. An estimated total number of 200 and 100 radiotherapeutic regimens with 18-20 Gy applied to cases with contralateral GCNis and with TSS, respectively, are documented in the literature. CONCLUSION: Cumulative experience suggests that radiotherapy with 18-20 Gy to the testis may fail with an estimated frequency of around 1%. Reasons for failure are elusive. A primary radioresistant subfraction of GCNis is hypothesized as well as technical failures regarding application of the radiotherapeutic dose volume in small and mobile testes. Caregivers of patients with TSS and contralateral GCNis should be aware of local relapses occurring after intervals of > 10 years.

The DNA methylation profile of human spermatogonia at single-cell- and single-allele-resolution refutes its role in spermatogonial stem cell function and germ cell differentiation.

Human spermatogonial stem cells (hSSCs) have potential in fertility preservation of prepubertal boys or in treatment of male adults suffering from meiotic arrest. Prior to therapeutic application, in vitro propagation of rare hSSCs is mandatory. As the published data points to epigenetic alterations in long-term cell culture of spermatogonia (SPG), an initial characterisation of their DNA methylation state is important. Testicular biopsies from five adult normogonadotropic patients were converted into aggregate-free cell suspensions. FGFR3-positive (FGFR3+) SPG, resembling a very early stem cell state, were labelled with magnetic beads and isolated in addition to unlabelled SPG (FGFR3-). DNA methylation was assessed by limiting dilution bisulfite pyrosequencing for paternally imprinted (H19 and MEG3), maternally imprinted (KCNQ1OT1, PEG3, and SNRPN), pluripotency (POU5F1/OCT4 and NANOG), and spermatogonial/hSSC marker (FGFR3, GFRA1, PLZF, and L1TD1) genes on either single cells or pools of 10 cells. Both spermatogonial subpopulations exhibited a methylation pattern largely equivalent to sperm, with hypomethylation of hSSC marker and maternally imprinted genes and hypermethylation of pluripotency and paternally imprinted genes. Interestingly, we detected fine differences between the two spermatogonial subpopulations, which were reflected by an inverse methylation pattern of imprinted genes, i.e. decreasing methylation in hypomethylated genes and increasing methylation in hypermethylated genes, from FGFR3+ through FGFR3- SPG to sperm. Limitations of this study are due to it not being performed on a genome-wide level and being based on previously published regulatory gene regions. However, the concordance of DNA methylation between SPG and sperm implies that hSSC regulation and germ cell differentiation do not occur at the DNA methylation level.

Dynamics, ultrastructure and gene expression of human in vitro organized testis cells from testicular sperm extraction biopsies.

STUDY QUESTION: Is it possible to induce in vitro reorganization of primary human testis cells from testicular sperm extraction (TESE) biopsies, maintain their long-term cultivation in a 2D system and identify cellular compositions? SUMMARY ANSWER: In vitro reorganization of primary human testis cells from TESE biopsies and their long-term cultivation on uncoated cell culture dishes is feasible and the cellular compositions can be uncovered through gene expression and microscopic analyses. WHAT IS KNOWN ALREADY: It has been shown in the rodent model that mixtures of testicular cell types are able to reassemble into clusters when cultivated on different kinds of surfaces or three-dimensional matrices. Two recent publications demonstrated the ability of primary human testicular cells to assemble into testicular organoids and their cultivation for a period of 3-4 weeks. STUDY DESIGN SIZE, DURATION: Primary human testis cells from TESE biopsies from 16 patients were reorganized in vitro and the clusters were cultivated long term on uncoated cell culture dishes, providing a solid ground for in vitro spermatogenesis. Gene expression analysis as well as fluorescence/transmission electron microscopy (TEM) were employed to uncover the cellular composition of the clusters. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsies from adult, normogonadotropic patients displaying full spermatogenesis (n = 11), hypospermatogenesis (n = 2), predominantly full spermatogenesis with some hypospermatogenic tubules (n = 1), meiotic arrest (n = 1) or mixed atrophy (n = 1) were enzymatically digested and dispersed cells were cultivated on 96-well plates or chamber dishes as aggregate-free cell suspensions. Time-lapse imaging of cluster formation was performed over a period of 48 h. For receptor tyrosine kinase inhibition of cluster formation, cells were treated twice with K252a within 2-3 days. Immunofluorescence staining and confocal microscopy was carried out on clusters after 1-3 weeks of cultivation to identify the presence of Sertoli cells (SC) (SOX9), peritubular myoid cells (SMA), Leydig cells (LC) (STAR), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Single clusters from four patients and a pool of eight larger clusters from another patient were manually picked and subjected to quantitative real-time PCR to evaluate the presence of SC (SOX9, AR), LC (INSL3, STAR, HSD3B1), peritubular myoid cells (ACTA2), fibroblasts (FSP1), endothelial cells (CD34), macrophages (CD68), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Finally, an ultrastructural investigation was conducted based on TEM of clusters from six different patients, among them 3-month cultivated large clusters from two patients. MAIN RESULTS AND THE ROLE OF CHANCE: Quantitative PCR-based analysis of single-picked testicular cell clusters identified SC, peritubular myoid cells, endothelial cells, fibroblasts, macrophages, spermatids and LC after 1, 2 or 3 weeks or 3 months of cultivation. Immunofluorescence positivity for SC and peritubular myoid cells corroborated the presence of these two kinds of testis niche cells. In addition, round as well as elongated spermatids were frequently encountered in 1 and 2 weeks old clusters. Transmission electron microscopical classification confirmed all these cell types together with a few spermatogonia. Macrophages were found to be of the proinflammatory M1 subtype, as revealed by CD68+/CD163-/IL6+ expression. Time-lapse imaging uncovered the specific dynamics of cluster fusion and enlargement, which could be prevented by addition of protein kinase inhibitor K252a. LARGE SCALE DATA: N/A. LIMITATIONS REASON FOR CAUTION: Cell composition of the clusters varied based on the spermatogenic state of the TESE patient. Although spermatids could be observed with all applied methods, spermatogonia were only detected by TEM in single cases. Hence, a direct maintenance of these germ cell types by our system in its current state cannot be postulated. Moreover, putative dedifferentiation and malignant degeneration of cells in long-term cluster cultivation needs to be investigated in the future. WIDER IMPLICATIONS OF THE FINDINGS: This work demonstrates that the reorganization of testicular cells can be achieved with TESE biopsies obtained from men enroled in a standard clinical assisted reproduction program. The formed clusters can be cultivated for at least 3 months and are composed, to a large extent, of the most important somatic cell types that are essential to support spermatogenesis. These findings may provide the cellular basis for advances in human in vitro spermatogenesis and/or the possibility for propagation of spermatogonia within a natural stem cell niche-like environment. STUDY FUNDING AND COMPETING INTERESTS: The project was funded by a DFG grant to K.v.K. (KO 4769/2-1). The authors declare they have no conflicts of interest.

Alcohol and male reproductive health: a cross-sectional study of 8344 healthy men from Europe and the USA.

STUDY QUESTION: Is there an association between alcohol intake and semen quality and serum reproductive hormones among healthy men from the USA and Europe? SUMMARY ANSWER: Moderate alcohol intake is not adversely associated with semen quality in healthy men, whereas it was associated with higher serum testosterone levels. WHAT IS KNOWN ALREADY: High alcohol intake has been associated with a wide range of diseases. However, few studies have examined the correlation between alcohol and reproductive function and most have been conducted in selected populations of infertile men or have a small sample size and the results have been contradictory. STUDY DESIGN, SIZE, DURATION: A coordinated international cross-sectional study among 8344 healthy men. A total of 1872 fertile men aged 18-45 years (with pregnant partners) from four European cities and four US states, and 6472 young men (most with unknown fertility) aged 18-28 years from the general population in six European countries were recruited. PARTICIPANTS/MATERIALS, SETTING, METHODS: The men were recruited using standardized protocols. A semen analysis was performed and men completed a questionnaire on health and lifestyle, including their intake of beer, wine and liquor during the week prior to their visit. Semen quality (semen volume, sperm concentration, percentage motile and morphologically normal sperm) and serum reproductive hormones (FSH, LH, testosterone, sex hormone-binding globulin, and inhibin B and free testosterone) were examined. MAIN RESULTS AND THE ROLE OF CHANCE: The participation rate for our populations was 20-30%. We found no consistent association between any semen variable and alcohol consumption, which was low/moderate in this group (median weekly intake 8 units), either for total consumption or consumption by type of alcohol. However, we found a linear association between total alcohol consumption and total or free testosterone in both groups of men. Young and fertile men who consumed >20 units of alcohol per week had, respectively, 24.6 pmol/l (95% confidence interval 16.3-32.9) and 19.7 pmol/l (7.1-32.2) higher free testosterone than men with a weekly intake between 1 and 10 units. Alcohol intake was not significantly associated with serum inhibin B, FSH or LH levels in either group of men. The study is the largest of its kind and has sufficient power to detect changes in semen quality and reproductive hormones. LIMITATIONS, REASONS FOR CAUTION: The participation rate was low, but higher than in most previous semen quality studies. In addition, the study was cross-sectional and the men were asked to recall their alcohol intake in the previous week, which was used as a marker of intake up to 3 months before. If consumption in that week differed from the typical weekly intake and the intake 3 months earlier, misclassification of exposure may have occurred. However, the men were unaware of their semen quality when they responded to the questions about alcohol intake. Furthermore, we cannot exclude that our findings are due to unmeasured confounders, including diet, exercise, stress, occupation and risk-taking behavior. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests that moderate alcohol intake is not adversely associated with semen quality in healthy men, whereas it was associated with higher serum testosterone levels which may be due to a changed metabolism of testosterone in the liver. Healthy men may therefore be advised that occasional moderate alcohol intake may not harm their reproductive health; we cannot address the risk of high alcohol consumption of longer duration or binge drinking on semen quality and male reproductive hormones. STUDY FUNDING/COMPETING INTERESTS: All funding sources were non-profitable and sponsors of this study played no role in the study design, in data collection, analysis, or interpretation, or in the writing of the article. The authors have no conflicts of interest.

Germ Cell Maintenance and Sustained Testosterone and Precursor Hormone Production in Human Prepubertal Testis Organ Culture with Tissues from Boys 7 Years+ under Conditions from Adult Testicular Tissue.

Human prepubertal testicular tissues are rare, but organ culture conditions to develop a system for human in vitro-spermatogenesis are an essential option for fertility preservation in prepubertal boys subjected to gonadotoxic therapy. To avoid animal testing in line with the 3Rs principle, organ culture conditions initially tested on human adult testis tissue were applied to prepubertal samples (n = 3; patient ages 7, 9, and 12 years). Tissues were investigated by immunostaining and transmission electron microscopy (TEM), and the collected culture medium was profiled for steroid hormones by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Culture conditions proved suitable for prepubertal organ culture since SSCs and germ cell proliferation could be maintained until the end of the 3-week-culture. Leydig cells (LCs) were shown to be competent for steroid hormone production. Three additional testis tissues from boys of the same age were examined for the number of germ cells and undifferentiated spermatogonia (SPG). Using TEM micrographs, eight tissues from patients aged 1.5 to 13 years were examined, with respect to the sizes of mitochondria (MT) in undifferentiated SPG and compared with those from two adult testicular tissues. Mitochondrial sizes were shown to be comparable between adults and prepubertal boys from approximately 7 years of age, which suggests the transition of SSCs from normoxic to hypoxic metabolism at about or before this time period.

Semen quality in sub-fertile range for a significant proportion of young men from the general German population: a co-ordinated, controlled study of 791 men from Hamburg and Leipzig.

Population studies have shown that a high proportion of Nordic men may have so poor semen quality that they can be classified as sub-fertile according to international standards. A question is whether the Nordic data are specific for the Nordic countries or they should be seen as an expression of a general trend in Europe. We therefore carried out a prospective study of semen quality of young men raised in the former East Germany (Leipzig) and West Germany (Hamburg). To enable inter-regional comparisons, we utilized a common European research protocol previously used in studies in the Nordic-Baltic region. Three hundred and thirty-four young men representative of the general population from Hamburg, and 457 from Leipzig delivered semen samples, underwent physical examinations and provided information on life-style and reproductive health parameters. The study period in Hamburg was February 2003--July 2004, and in Leipzig July 2003--April 2005. No significant differences were observed in sperm concentration (median 46, 42, and 44 million/mL for men from Hamburg, Leipzig and the combined Hamburg-Leipzig group respectively) or total sperm count (154,141 and 149 million), whereas the differences for morphologically normal spermatozoa (9.4 and 8.4%) and motile spermatozoa (67 and 81%) were significantly different. Previously published studies have shown reduced fertility with decreasing sperm concentrations below 40-55 millions/mL and normal sperm morphology below 9-19%. Thus, a large fraction of young German men seem to have impaired semen quality that may reduce their natural fertility. However, it remains to be investigated to what extent poor semen quality contributes to the low German fertility rates.

Varicocele Is Associated with Impaired Semen Quality and Reproductive Hormone Levels: A Study of 7035 Healthy Young Men from Six European Countries.

BACKGROUND: Present knowledge on the impact of varicoceles on testicular function is largely based on studies of subfertile and infertile men, making it difficult to extrapolate the impact of varicocele on the general population. OBJECTIVE: To describe associations between varicocele and testicular function assessed by semen analysis and reproductive hormones in men from the general population. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional multicentre study of 7035 young men, median age 19 yr, from the general population in six European countries (Denmark, Finland, Germany, Estonia, Latvia, and Lithuania) were investigated from 1996 to 2010. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We analysed results from physical examination, conventional semen variables, and serum reproductive hormones using multivariable regression analyses. RESULTS AND LIMITATIONS: A total of 1102 (15.7%) had grade 1-3 varicocele. Increasing varicocele grade was associated with poorer semen quality, even in grade 1 varicocele. In grade 3 varicocele, sperm concentration was less than half of that in men with no varicocele. Presence of varicocele was also associated with higher serum levels of follicle-stimulating hormone, lower inhibin B, and higher levels of luteinising hormone; testosterone and free testosterone were not significantly different between men with and without varicocele. This study cannot draw a conclusion on the progressiveness of varicocele or the effect of treatment. CONCLUSIONS: We demonstrated an adverse effect of increasing grade of varicocele on testicular function in men not selected due to fertility status. PATIENT SUMMARY: The presence and increasing grade of varicocele is adversely associated with semen quality and reproductive hormone levels in young men from the general population.